Although many theories of learning have proposed that plasticity at specific synapses in the central nervous system is critical to memory storage, there have been very few experimental preparations in which it has been possible to test this hypothesis rigorously. For this reason, the past program of research supported by this grant has been devoted to studies of the cellular mechanisms of simple forms of learning of the gill-and siphon-withdrawal reflex in Aplysia. Previous has suggested that a mechanism to dishabituation and sensitization of the reflex is presynaptic facilitation of the siphon sensory neurons, and that a mechanism contribution to classical conditioning of the reflex is an amplification of this mechanism by sensitization by the occurrence of spike activity in the sensory neurons just before the sensitizing stimulus. Recent results have suggested that in addition to this presynaptic mechanism, Hebbian long-term potentiation (LTP) may also contribute to conditioning of the reflex. However, the studies performed at the behavioral and cellular levels have generally used different preparations and procedures, so that the relationship between the two was inferred rather than tested directly. To test the relationship between cellular events and behavior more rigorously, we developed two simplified preparations with which it is relatively easy to record the activity of single identified neurons during behavior. Our initial studies with these preparations provided the first direct evidence that habituation, dishabituation, and sensitization of the reflex involve plasticity at the sensory-motor neuron synapses. In addition, our results have indicated that other sites and mechanisms of plasticity also contribute. Thus, these preparations provide an opportunity to analyze parallel processing and examine mechanisms contribution to learning at the system level as well as the cellular level. We now propose to utilize these simplified preparations to examine in more detail the mechanisms contribution to dishabituation and sensitization, and to perform a similar analysis of classical conditioning. Specifically, we will test the contributions of activity-dependent presynaptic facilitation and Hebbian long-term potentiation to conditioning, differential conditioning, and second-order conditioning, and also examine the contribution of their cellular mechanisms and explore how they are integrated at the behavioral level. In addition to these studies attempting to relate cellular events to behavior, we also plan to perform experiments on single sensory and motor neurons in cell culture to analyze mechanisms of activity-dependent plasticity (post-tetanic potentiation, activity-dependent facilitation by serotonin, and LTP) that may contribute to the learning.